Spironolactone: stability indicating method for the identification of major degradation products and pathways
AGENCE GÉNÉRALE DES ÉQUIPEMENTS ET PRODUITS DE SANTÉ
Legrand P1,2, Ribier Z1, Stevenin J1, Boccadifuoco G1, Wojcicki A1, Boudy V 1,2, Goulay-Dufaÿ S1 1: Agence Générale des Equipements et Produits de Santé (AGEPS), Assistance Publique – Hôpitaux de Paris (AP-HP), France
2: Unité de Technologies Chimiques et Biologiques pour la Santé (UMR8258, Inserm U1022), Faculty of Pharmacy, Paris Descartes University, France
Introduction
spironolactone 7 α thio spironolactone canrenone
Fig.1 : Major degradation pathway of spironolactone, via alkaline hydrolisis
Spironolactone is a potassium-sparing diuretic, used in various
diseases such as high blood pressure or hyperaldosteronism. In
this study, we developed a stability indicating method and
performed a forced degradation study on this molecule to identify
major degradation pathways and degradation products of
spironolactone during long term stability study.
Material & Method
Final chromatographic conditions
• Column: Luna phenyl hexyl (3 µm,
150 × 4.6 mm)
• Mobile phase: 63 water / 30
acetonitrile / 7 methanol (v/v/v)
• Time of run: 55 min
• flow rate of 1.5 mL/min
• Wavelenght: 254 nm and scan rate
Forced degradation study
Objectifs
• Approximatively 15 % of degradation
• Pure peak of spironolactone
• Pure peak of the majors impurities
• Mass balance conserved
Forced degradation conditions
Type of stress conditions
Temperature 60°C – 2 hours
photodegradation UV lamp – 48 hours
oxidation H2O2 30 % - 24 hours
Acidic HCl 0,1 M – 24 hours
Basic NaOH 0,01 M – 15 minutes
Neutral pH 5 days
Results & Discussion
Type of stress conditions
degradation of
spironolactone
(%)
Sum of
impurities (%)
Pure peak of
spironolactone / of
the impurities
Temperature 60°C – 2 hours 15 10 Yes / yes
photodegradatio
n UV lamp – 48 hours 7 0,4 Yes / yes
oxidation H2O2 30 % - 24
hours 12 2
Yes / yes
Acidic HCl 0,1 M – 48 hours 15 12 Yes / yes
Basic NaOH 0,01 M – 15
minutes 11 10
Yes / yes
Neutral pH 5 days 12 11 Yes / yes
Table 1. Results of the forced degradation study
Forced degradation conditions were
optimized to avoid more than 15 % of
spironolactone degradation, in order to
limit secondary degradations.
Mass balance is approximatively
conserved in the case of stress induced
by temperature and acidic neutral or
basic conditions. Canrenone, one of the
major degradation impurity have a
maximum of absorption at 280 nm which
can explain the small differences in mass
balances observed
For all type of stress, the chromatographic peaks are pure
0,0 2,5 5,0 7,5 10,0 12,5 15,0 17,5 20,0 22,5 25,0 27,5 30,0 32,5 35,0 37,5 40,0 42,5 45,0 47,5 50,0 52,5 55,0
-20
0
20
40
60
80
100
120
140
160
180
Time (min)
Inte
nsity (
mA
U)
Fig.2 : Chromatogram of spironolactone (1 mg/mL) without stress (black line) and after 15 minutes with NaOH 0,01M (red line)
1
2
1
2
3
1 : canrenone
2 : 7 α thio spironolactone
3 : spironolactone
* : unidentified degradation
products
The two main degradation products : canrenone and 7 α
thio spironolactone are well separated (Rs > 1,5).
The LOD and the LOQ (0,3 µg/mL and 0,9 µg/mL
respectively) enable the detection of impurity at 0,05 % of
spironolactone (ICH recommandations).
* *
In case of stress induced by oxidation
(H2O2 and photooxidation), the mass
balance is incorrect: the degradation
products are not observed via the
developped methd. But oxidation appears
not to be the major degradation patway
Some degradation products formed in stressed conditions
are observed but not identify yet. If these impurities are
observed during stability studies, there identification will
be needed.
Conclusion The stability indicating method developed
allows the elution of spironolactone as a
spectrally pure peak, and well resolved
from its degradation products. The two
main degradation products are well
separated (RS>1,5) allowing their
evaluation during long term study.